Troponin C (TnC) is implicated in the initiation of myocyte contraction via binding of cytosolic and subsequent recognition of the Troponin I switch peptide. Mutations of the cardiac TnC N-terminal regulatory domain have been shown to alter both calcium binding and myofilament force generation. We have performed molecular dynamics simulations of engineered TnC variants that increase or decrease sensitivity, in order to understand the structural basis of their impact on TnC function. We will use the distinction for mutants that are associated with increased affinity and for those mutants with reduced affinity. Our studies demonstrate that for GOF mutants V44Q and L48Q, the structure of the physiologically-active site II binding site in the -free (apo) state closely resembled the -bound (holo) state. In contrast, site II is very labile for LOF mutants E40A and V79Q in the apo form and bears little resemblance with the holo conformation. We hypothesize that these phenomena contribute to the increased association rate, , for the GOF mutants relative to LOF. Furthermore, we observe significant positive and negative positional correlations between helices in the GOF holo mutants that are not found in the LOF mutants. We anticipate these correlations may contribute either directly to affinity or indirectly through TnI association. Our observations based on the structure and dynamics of mutant TnC provide rationale for binding trends observed in GOF and LOF mutants and will guide the development of inotropic drugs that target TnC.

Author Summary

Muscle cells contract using a network of thread-like protein assemblies called myofilaments. Contraction is preceded by a signal that causes calcium to rush into the cell cytosol, where it can freely diffuse to and bind the myofilament proteins. Troponin C, a calcium sensor located on the thin filament, initiates and regulates the cascade of changes resulting in the generation of force by the thin and thick filaments comprising the myofilament lattice. In heart tissue, pathological conditions known as dilated and hypertrophic cardiomyopathies (DCM and HCM, respectively) are in part associated with abnormalities in the ability of the myofilaments to generate force at normal calcium concentrations. Manipulation of Troponin C calcium-binding through protein engineering and pharmaceutical intervention has thus attracted considerable attention as a therapeutic strategy for ameliorating these cardiac defects. In this study, we uncover a molecular basis of altered calcium handling for several engineered Troponin C variants, which provides further insight into tuning its control of myofilament contraction.

Highlights

► ACh and ET-1 activate a K+ current in cardiac atrioventricular nodal cells. ► Tertiapin-Q sensitive IKACh activated via M2 receptors shows bi-exponential ‘fade’. ► ET-1 activates a similar current that also fades. ► The fade reflects desensitization rather than altered K+ ion driving force. ► Acetylcholine is able to cross-desensitize the AVN cell response to endothelin-1.

The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, IKACh. However, it is not yet known if this response exhibits ‘fade’ or desensitization in the AVN, as established for the heart’s primary pacemaker – the sinoatrial node. In this study, acute activation of IKACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37 °C. 0.1–1 μM acetylcholine (ACh) rapidly activated a robust IKACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300 nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1 μM). During sustained ACh exposure the elicited IKACh exhibited bi-exponential fade (τf of 2.0 s and τs 76.9 s at −120 mV; 1 μM ACh). 10 nM ET-1 elicited a current similar to IKACh, which faded with a mono-exponential time-course (τ of 52.6 s at −120 mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization.

Selected abstracts delivered at the 9th Annual AOSpine North America Fellows Forum

Consistent with EBSJ's commitment to fostering quality research, we are pleased to feature some of the most highly rated abstracts from the 9th Annual AOSpine North America Fellows Forum in Banff, Canada. Enhancing the quality of evidence in spine care means acknowledging and supporting the efforts of young researchers within our AOSpine North America network. We look forward to seeing more from these promising researchers in the future.

Background

The atrioventricular node (AVN) is a key component of the cardiac pacemaker-conduction system. Although it is known that receptors for the peptide hormone endothelin-1 (ET-1) are expressed in the AVN, there is very little information available on the modulatory effects of ET-1 on AVN electrophysiology. This study characterises for the first time acute modulatory effects of ET-1 on AVN cellular electrophysiology.

Methods

Electrophysiological experiments were conducted in which recordings were made from rabbit isolated AVN cells at 35–37°C using the whole-cell patch clamp recording technique.

Results

Application of ET-1 (10 nM) to spontaneously active AVN cells led rapidly (within ∼13 s) to membrane potential hyperpolarisation and cessation of spontaneous action potentials (APs). This effect was prevented by pre-application of the ETA receptor inhibitor BQ-123 (1 µM) and was not mimicked by the ETB receptor agonist IRL-1620 (300 nM). In whole-cell voltage-clamp experiments, ET-1 partially inhibited L-type calcium current (ICa,L) and rapid delayed rectifier K+ current (IKr), whilst it transiently activated the hyperpolarisation-activated current (If) at voltages negative to the pacemaking range, and activated an inwardly rectifying current that was inhibited by both tertiapin-Q (300 nM) and Ba2+ ions (2 mM); each of these effects was sensitive to ETA receptor inhibition. In cells exposed to tertiapin-Q, ET-1 application did not produce membrane potential hyperpolarisation or immediate cessation of spontaneous activity; instead, there was a progressive decline in AP amplitude and depolarisation of maximum diastolic potential.

Conclusions

Acutely applied ET-1 exerts a direct modulatory effect on AVN cell electrophysiology. The dominant effect of ET-1 in this study was activation of a tertiapin-Q sensitive inwardly rectifying K+ current via ETA receptors, which led rapidly to cell quiescence.

Depolarization of an individual mitochondrion or small clusters of mitochondria within cells has been achieved using a photoactivatable probe. The probe is targeted to the matrix of the mitochondrion by an alkyltriphenylphosphonium lipophilic cation and releases the protonophore 2,4-dinitrophenol locally in predetermined regions in response to directed irradiation with UV light via a local photolysis system. This also provides a proof of principle for the general temporally and spatially controlled release of bioactive molecules, pharmacophores, or toxins to mitochondria with tissue, cell, or mitochondrion specificity.

We consider the design of an effective and reliable adaptive finite element method (AFEM) for the nonlinear Poisson-Boltzmann equation (PBE). We first examine the two-term regularization technique for the continuous problem recently proposed by Chen, Holst, and Xu based on the removal of the singular electrostatic potential inside biomolecules; this technique made possible the development of the first complete solution and approximation theory for the Poisson-Boltzmann equation, the first provably convergent discretization, and also allowed for the development of a provably convergent AFEM. However, in practical implementation, this two-term regularization exhibits numerical instability. Therefore, we examine a variation of this regularization technique which can be shown to be less susceptible to such instability. We establish a priori estimates and other basic results for the continuous regularized problem, as well as for Galerkin finite element approximations. We show that the new approach produces regularized continuous and discrete problems with the same mathematical advantages of the original regularization. We then design an AFEM scheme for the new regularized problem, and show that the resulting AFEM scheme is accurate and reliable, by proving a contraction result for the error. This result, which is one of the first results of this type for nonlinear elliptic problems, is based on using continuous and discrete a priori L∞ estimates to establish quasi-orthogonality. To provide a high-quality geometric model as input to the AFEM algorithm, we also describe a class of feature-preserving adaptive mesh generation algorithms designed specifically for constructing meshes of biomolecular structures, based on the intrinsic local structure tensor of the molecular surface. All of the algorithms described in the article are implemented in the Finite Element Toolkit (FETK), developed and maintained at UCSD. The stability advantages of the new regularization scheme are demonstrated with FETK through comparisons with the original regularization approach for a model problem. The convergence and accuracy of the overall AFEM algorithm is also illustrated by numerical approximation of electrostatic solvation energy for an insulin protein.

Study design: Prospective randomized animal study.

Objective: To determine a surgical technique for reproducible and functional intervertebral disc replacement in an orthotopic animal model.

Methods: The caudal 3/4 intervertebral disc (IVD) of the rat tail was approached by two surgical techniques: blunt dissection, stripping and retracting (Technique 1) or incising and repairing (Technique 2) the dorsal longitudinal tendons. The intervertebral disc was dissected and removed, and then either discarded or reinserted. Outcome measures were perioperative complications, spontaneous tail movement, 7T MRI (T1- and T2-sequences for measurement of disc space height (DSH) and disc hydration). Microcomputed tomographic imaging (micro CT) was additionally performed postmortem.

Results: No vascular injuries occurred and no systemic or local infections were observed over the course of 1 month. Tail movements were maintained. With tendon retraction (Technique 1) gross loss of DSH occurred with both discectomy and reinsertion. Tendon division (Technique 2) maintained DSH with IVD reinsertion but not without. The DSH was demonstrated on MRI measurement. A new scoring system to assess IVD appearances was described.

Conclusions: The rat tail model, with a tendon dividing surgical technique, can function as an orthotopic animal model for IVD research. Mechanical stimulation is maintained by preserved tail movements. 7T MRI is a feasible modality for longitudinal monitoring for the rat caudal disc.

Background

The realm of pathological entities can be subdivided into pathological dispositions, pathological processes, and pathological structures. The latter are the bearer of dispositions, which can then be realized by their manifestations — pathologic processes. Despite its ontological soundness, implementing this model via purpose-oriented domain ontologies will likely require considerable effort, both in ontology construction and maintenance, which constitutes a considerable problem for SNOMED CT, presently the largest biomedical ontology.

Results

We describe an ontology design pattern which allows ontologists to make assertions that blur the distinctions between dispositions, processes, and structures until necessary. Based on the domain upper-level ontology BioTop, it permits ascriptions of location and participation in the definition of pathological phenomena even without an ontological commitment to a distinction between these three categories. An analysis of SNOMED CT revealed that numerous classes in the findings/disease hierarchy are ambiguous with respect to process vs. disposition. Here our proposed approach can easily be applied to create unambiguous classes. No ambiguities could be defined regarding the distinction of structure and non-structure classes, but here we have found problematic duplications.

Conclusions

We defend a judicious use of disjunctive, and therefore ambiguous, classes in biomedical ontologies during the process of ontology construction and in the practice of ontology application. The use of these classes is permitted to span across several top-level categories, provided it contributes to ontology simplification and supports the intended reasoning scenarios.

Investigations into the functional modulation of the cardiac Na+–Ca2+ exchanger (NCX) by acute β-adrenoceptor/PKA stimulation have produced conflicting results. Here, we investigated (i) whether or not β-adrenoceptor activation/PKA stimulation activates current in rabbit cardiac myocytes under NCX-‘selective’ conditions and (ii) if so, whether a PKA-activated Cl−-current may contribute to the apparent modulation of NCX current (INCX). Whole-cell voltage-clamp experiments were conducted at 37 °C on rabbit ventricular and atrial myocytes. The β-adrenoceptor-activated currents both in NCX-‘selective’ and Cl−-selective recording conditions were found to be sensitive to 10 mM Ni2+. In contrast, the PKA-activated Cl− current was not sensitive to Ni2+, when it was activated downstream to the β-adrenoceptors using 10 μM forskolin (an adenylyl cyclase activator). When 10 μM forskolin was applied under NCX-selective recording conditions, the Ni2+-sensitive current did not differ between control and forskolin. These findings suggest that in rabbit myocytes: (a) a PKA-activated Cl− current contributes to the Ni2+-sensitive current activated via β-adrenoceptor stimulation under recording conditions previously considered selective for INCX; (b) downstream activation of PKA does not augment Ni2+-sensitive INCX, when this is measured under conditions where the Ni2+-sensitive PKA-activated Cl− current is not present.

This paper is about how cortical recurrent interactions in primary visual cortex (V1) together with feedback from extrastriate cortex can account for spectral peaks in the V1 local field potential (LFP). Recent studies showed that visual stimulation enhances the γ-band (25–90 Hz) of the LFP power spectrum in macaque V1. The height and location of the γ-band peak in the LFP spectrum were correlated with visual stimulus size. Extensive spatial summation, possibly mediated by feedback connections from extrastriate cortex and long-range horizontal connections in V1, must play a crucial role in the size dependence of the LFP. To analyze stimulus-effects on the LFP of V1 cortex, we propose a network model for the visual cortex that includes two populations of V1 neurons, excitatory and inhibitory, and also includes feedback to V1 from extrastriate cortex. The neural network model for V1 was a resonant system. The model’s resonance frequency (ResF) was in the γ-band and varied up or down in frequency depending on cortical feedback. The model’s ResF shifted downward with stimulus size, as in the real cortex, because increased size recruited more activity in extrastriate cortex and V1 thereby causing stronger feedback. The model needed to have strong local recurrent inhibition within V1 to obtain ResFs that agree with cortical data. Network resonance as a consequence of recurrent excitation and inhibition appears to be a likely explanation for γ-band peaks in the LFP power spectrum of the primary visual cortex.

Protein cysteine residues are central to redox signaling and to protection against oxidative damage through their interactions with reactive oxygen and nitrogen species, and electrophiles. Although there is considerable evidence for a functional role for cysteine modifications, the identity and physiological significance of most protein thiol alterations are unknown. One way to identify candidate proteins involved in these processes is to utilize the proteomic methodologies that have been developed in recent years for the identification of proteins that undergo cysteine modification in response to redox signals or oxidative damage. These tools have proven effective in uncovering novel protein targets of redox modification and are important first steps that allow for a better understanding of how reactive molecules may contribute to signaling and damage. Here, we discuss a number of these approaches and their application to the identification of a variety of cysteine-centered redox modifications.

The aim of the study was to determine whether the incidence of radiolucencies can be reduced using pulsed lavage before cementing the tibia in unicompartmental knee arthroplasty (UKA). We prospectively studied a consecutive series of 112 cemented Oxford UKA in 100 patients in two centres. In group A (n = 56) pulsed lavage and in group B (n = 56) conventional syringe lavage was used to clean the cancellous bone. The same standardised cementing technique was applied in all cases. At a minimum follow-up of one year patients were evaluated clinically and screened radiographs were obtained. The cement bone interface under the tibial plateau was divided into four zones and evaluated for the presence of radiolucent lines. All radiographs were evaluated (n = 112), and radiolucencies in all four zones were found in two cases in group A (4%) and in 12 cases in group B (22%) (p = 0.0149). Cement penetration showed a median of 2.6 mm (group A) and 1.5 mm (group B) (p < 0.0001). We recommend the routine use of pulsed lavage in Oxford UKA to reduce the incidence of radiolucency and to improve long-term fixation.

Objectives

To review the Yorkshire Laser Centre experience with bronchoscopic photodynamic therapy (PDT) in early central lung cancer in subjects not eligible for surgery and to discuss diagnostic problems and the indications for PDT in such cases.

Methods

Of 200 patients undergoing bronchoscopic PDT, 21 had early central lung cancer and were entered into a prospective study. Patients underwent standard investigations including white light bronchoscopy in all and autofluorescence bronchoscopy in 12 of the most recent cases. Indications for bronchoscopic PDT were recurrence/metachronous endobronchial lesions following previous treatment with curative intent in 10 patients (11 lesions), ineligibility for surgery because of poor cardiorespiratory function in 8 patients (9 lesions) and declined consent to operation in 3 patients. PDT consisted of intravenous administration of Photofrin 2 mg/kg followed by bronchoscopic illumination 24–48 h later.

Results

29 treatments were performed in 21 patients (23 lesions). There was no procedure‐related or 30 day mortality. One patient developed mild skin photosensitivity. All patients expressed satisfaction with the treatment and had a complete response of variable duration. Six patients died at 3–103 months (mean 39.3), three of which were not as a result of cancer. Fifteen patients were alive at 12–82 months.

Conclusion

Bronchoscopic PDT in early central lung cancer can achieve long disease‐free survival and should be considered as a treatment option in those ineligible for resection. Autofluorescence bronchoscopy is a valuable complementary investigation for identification of synchronous lesions and accurate illumination in bronchoscopic PDT.

The haploid microspore division during pollen development in flowering plants is an intrinsically asymmetric division which establishes the male germline for sexual reproduction. Arabidopsis gem1 mutants lack the male germline as a result of disturbed microspore polarity, division asymmetry, and cytokinesis and represent loss-of-function mutants in MOR1/GEM1, a plant orthologue of the conserved MAP215/Dis1 microtubule associated protein (MAP) family. This provides genetic evidence for the role of MAP215/Dis1 in the organization of gametophytic microtubule arrays, but it has remained unknown how microtubule arrays are affected in gem1 mutant microspores. Here, novel male gametophytic microtubule-reporter Nicotiana tabacum plants were constructed, expressing a green fluorescent protein-α-TUBULIN fusion protein (GFP-TUA6) under the control of a microspore-specific promoter. These plants allow effective visualization of all major male gametophytic microtubule arrays and provide useful tools to study the regulation of microtubule arrays by MAPs and other effectors. Depletion of TMBP200, a tobacco homologue of MOR1/GEM1 in gametophytic microtubule-reporter plants using microspore-targeted RNA interference, induced defects in microspore polarity, division asymmetry and cytokinesis that were associated with striking defects in phragmoplast position, orientation, and structure. Our observations further reveal a requirement for TMBP200 in gametophytic spindle organization and a novel role in spindle position and orientation in polarized microspores. These results provide direct evidence for the function of MAP215/Dis1 family protein TMBP200 in the organization of microtubule arrays critical for male germline formation in plants.

Background

We developed and tested a new method, called the Evidence-based Practice for Improving Quality method, for continuous quality improvement.

Methods

We used cluster randomization to assign 6 neonatal intensive care units (ICUs) to reduce nosocomial infection (infection group) and 6 ICUs to reduce bronchopulmonary dysplasia (pulmonary group). We included all infants born at 32 or fewer weeks gestation. We collected baseline data for 1 year. Practice change interventions were implemented using rapid-change cycles for 2 years.

Results

The difference in incidence trends (slopes of trend lines) between the ICUs in the infection and pulmonary groups was − 0.0020 (95% confidence interval [CI] − 0.0007 to 0.0004) for nosocomial infection and − 0.0006 (95% CI − 0.0011 to − 0.0001) for bronchopulmonary dysplasia.

Interpretation

The results suggest that the Evidence-based Practice for Improving Quality method reduced bronchopulmonary dysplasia in the neonatal ICU and that it may reduce nosocomial infection.

Nav1.5 is the principal voltage-gated sodium channel expressed in heart, and is also expressed at lower abundance in embryonic dorsal root ganglia (DRG) with little or no expression reported postnatally. We report here the expression of Nav1.5 mRNA isoforms in adult mouse and rat DRG. The major isoform of mouse DRG is Nav1.5a, which encodes a protein with an IDII/III cytoplasmic loop reduced by 53 amino acids. Western blot analysis of adult mouse DRG membrane proteins confirmed the expression of Nav1.5 protein. The Na+ current produced by the Nav1.5a isoform has a voltage-dependent inactivation significantly shifted to more negative potentials (by ~5 mV) compared to the full-length Nav1.5 when expressed in the DRG neuroblastoma cell line ND7/23. These results imply that the alternatively spliced exon 18 of Nav1.5 plays a role in channel inactivation and that Nav1.5a is likely to make a significant contribution to adult DRG neuronal function.

Impairments in feedback processing and reinforcement learning appear to be prominent aspects of schizophrenia (SZ), which may relate to symptoms of the disorder. Evidence from cognitive neuroscience investigations indicates that disparate brain systems may underlie different kinds of feedback-driven learning. The ability to rapidly shift response tendencies in the face of negative feedback, when reinforcement contingencies are reversed, is an important type of learning thought to depend on ventral prefrontal cortex (PFC). Schizophrenia has long been associated with dysfunction in dorsolateral areas of PFC, but evidence for ventral PFC impairment in more mixed. In order to assess whether SZ patients experience particular difficulty in carrying out a cognitive function commonly linked to ventral PFC function, we administered to 34 patients and 26 controls a modified version of an established probabilistic reversal learning task from the experimental literature (Cools et al. 2002). Although SZ patients and controls performed similarly on the initial acquisition of probabilistic contingencies, patients showed substantial learning impairments when reinforcement contingencies were reversed, achieving significantly fewer reversals [Χ^2(6)=15.717, p=0.008]. Even when analyses were limited to subjects who acquired all probabilistic contingencies initially (22 patients and 20 controls), patients achieved significantly fewer reversals [Χ^2(3)=9.408, p=0.024]. These results support the idea that ventral PFC dysfunction is a prevalent aspect of schizophrenic pathophysiology, which may contribute to deficits in reinforcement learning exhibited by patients. Further studies are required to investigate the roles of dopaminergic systems in these impairments.

Background

The effective functional analysis of male gametophyte development requires new tools enabling the spatially and temporally controlled expression of both marker genes and modified genes of interest. In particular, promoters driving expression at earlier developmental stages including microspores are required.

Results

Transcriptomic datasets covering four progressive stages of male gametophyte development in Arabidopsis were used to select candidate genes showing early expression profiles that were male gametophyte-specific. Promoter-GUS reporter analysis of candidate genes identified three promoters (MSP1, MSP2, and MSP3) that are active in microspores and are otherwise specific to the male gametophyte and tapetum. The MSP1 and MSP2 promoters were used to successfully complement and restore the male transmission of the gametophytic two-in-one (tio) mutant that is cytokinesis-defective at first microspore division.

Conclusion

We demonstrate the effective application of MSP promoters as tools that can be used to elucidate gametophytic gene functions in microspores in a male-specific manner.

Adult dragonflies augment their compound eyes with three simple eyes known as the dorsal ocelli. While the ocellar system is known to mediate stabilizing head reflexes during flight, the ability of the ocellar retina to dynamically resolve the environment is unknown. For the first time, we directly measured the angular sensitivities of the photoreceptors of the dragonfly median (middle) ocellus. We performed a second-order Wiener Kernel analysis of intracellular recordings of light-adapted photoreceptors. These were stimulated with one-dimensional horizontal or vertical patterns of concurrent UV and green light with different contrast levels and at different ambient temperatures. The photoreceptors were found to have anisotropic receptive fields with vertical and horizontal acceptance angles of 15° and 28°, respectively. The first-order (linear) temporal kernels contained significant undershoots whose amplitudes are invariant under changes in the contrast of the stimulus but significantly reduced at higher temperatures. The second-order kernels showed evidence of two distinct nonlinear components: a fast acting self-facilitation, which is dominant in the UV, followed by delayed self- and cross-inhibition of UV and green light responses. No facilitatory interactions between the UV and green light were found, indicating that facilitation of the green and UV responses occurs in isolated compartments. Inhibition between UV and green stimuli was present, indicating that inhibition occurs at a common point in the UV and green response pathways. We present a nonlinear cascade model (NLN) with initial stages consisting of separate UV and green pathways. Each pathway contains a fast facilitating nonlinearity coupled to a linear response. The linear response is described by an extended log-normal model, accounting for the phasic component. The final nonlinearity is composed of self-inhibition in the UV and green pathways and inhibition between these pathways. The model can largely predict the response of the photoreceptors to UV and green light.

Summary

Consumers of online health information are concerned with issues of quality and trust.

No sites presently offer comprehensive child health information and tools for families seeking solutions to complex questions that may involve disease, lifestyle, behavioral, and educational issues. Parents of children with complex health issues as well as parents of typically developing children, need a trusted, comprehensive online resource to inform and guide. To meet this need, The Hospital for Sick Children, with the support of founding sponsor TD Securities, launched a unique initiative, AboutKidsHealth in June2004. The project employs web technology combined with social marketing campaigns to promote and deliver evidence-based information and programmes in all major areas influencing child health and family quality of life. The web-based infrastructure will also be used to support selected research projects, and to provide enhanced communication and services for families of children with complex conditions and health professionals.

Late anemia is a well-recognized complication of Rhesus hemolytic disease of the newborn (HDN). The incidence of Rhesus HDN is declining, with a tendency for more severely affected pregnancies to be managed in specialist centres. Consequently, many paediatric departments may see relatively few affected infants with comparatively mild disease, and the risk of late anemia in such cases may not always be appreciated. Two cases of infants born with evidence of Rhesus isoimmunization noted at birth and encountering no immediate problems other than mild hyperbilirubinemia are described. After an uneventful early neonatal course, both infants were discharged without follow-up and presented in the second to third weeks of life with severe, life-threatening anemia, leading to neurological sequelae in one case. The importance of close surveillance, including hemoglobin measurements, in all infants with Rhesus hemolytic disease, irrespective of initial severity, is reiterated.